This is a competitive renewal for R01 NS39156, which supports our studies of Narp (Neuronal activity-regulated pentraxin). Narp is a secreted immediate early gene and is enriched at excitatory synapses where it binds and clusters AMPA-type glutamate receptors. Studies over the past 4 years have defined distinct protein determinants of the Narp for clustering on the membrane surface and interaction with AMPA type glutamate receptors. Based on an understanding of its protein chemistry, we generated dominant negative forms of Narp that cause rapid and selective degradation of native Narp in neurons, and used this tool to demonstrate an essential role of Narp in synaptogenesis. Ongoing studies indicate that Narp is co-functional with two other neural pentraxins, termed NP1 and NPR. Each pentraxin appears to contribute distinct features to a novel complex that forms during biosynthesis in which all three neural pentraxins are linked by disulfide bonds. Each pentraxin confers distinct properties on the heterocomplex. Narp confers the ability to form large clusters on the surface of cells, while NPR appears to function as a regulated membrane anchor. Aim 1 will define the molecular basis for co-assembly of these pentraxins and their clustering on the cell surface. Studies will examine the hypothesis that the N-terminal half is essential for a novel quaternary structure, which is dependent on specific intermolecular disulfide linkages. The role of NPR will be examined in detail. Aim 2 will extend ongoing studies of the interaction between Narp and AMPAR. Here the focus is on the C-terminal pentraxin domain, which mediates a direct but complex interaction between Narp and AMPAR. Studies will include efforts to generate and resolve a co-crystal. Aim 3 will examine mechanisms that mediate secretion of pentraxins from neurons, and test the hypothesis that Narp is packaged and secreted from a novel vesicle. These studies will address the important question of how pentraxins may be targeted by activity to specific synapses. Aim 4 will generate transgenic mice that conditionally express a GFP-tagged version of Narp that will facilitate analysis of secretion at mature synapses. We will also generate a second mouse model that conditionally expresses a dominant negative pentraxin that interrupts biosynthesis of Narp/NP1/NPR. These models will permit studies of the role of pentraxins in synaptic plasticity in the mature brain and test the hypothesized contribution of neural pentraxins to long-term plasticity.